Introduction: Osteophytes are a prominent feature of osteoarthritis (OA) joints and one of the clinical hallmarks of the disease progression. Research on osteophytes is fragmentary and modes of its contribution to OA pathology are obscure. Aim: To elucidate the role of osteophytes in OA pathology from a perspective of molecular and cellular events. Methods: RNA-seq of fully grown osteophytes, collected from tibial plateau of six OA patients revealed patterns corresponding to active extracellular matrix re-modulation and prominent participation of mast cells. Presence of mast cells was further confirmed by immunohistochemistry, performed on the sections of the osteophytes using anti-tryptase alpha/beta-1 and anti-FC epsilon RI antibodies and the related key up-regulated genes were validated by qRT-PCR. To test the role of OA synovial fluid (SF) in mast cell maturation as proposed by the authors, hematopoietic stem cells (HSCs) and ThP1 cells were cultured in a media supplemented with 10% SF samples, obtained from various grades of OA patients and were monitored using specific cell surface markers by flow cytometry. Proteomics analysis of SF samples was performed to detect additional markers specific to mast cells and inflammation that drive the cell differentiation and maturation. Results: Transcriptomics of osteophytes revealed a significant upregulation of mast cells specific genes such as chymase 1 (CMA1; 5-fold) carboxypeptidase A3 (CPA3; 4-fold), MS4A2/FCERI (FCERI; 4.2-fold) and interleukin 1 receptor-like 1 (IL1RL1; 2.5-fold) indicating their prominent involvement. (In IHC, anti-tryptase alpha/beta-1 and anti- FC epsilon RI-stained active mast cells were seen populated in cartilage, subchondral bone, and trabecular bone.) Based on these outcomes and previous learnings, the authors claim a possibility of mast cells invasion into osteophytes is mediated by SF and present in vitro cell differentiation assay results, wherein ThP1 and HSCs showed differentiation into HLA-DR+/CD206+ and FCERI+ phenotype, respectively, after exposing them to medium containing 10% SF for 9 days. Proteomics analysis of these SF samples showed an accumulation of mast cell-specific inflammatory proteins. Conclusions: RNA-seq analysis followed by IHC study on osteophyte samples showed a population of mast cells resident in them and may further accentuate inflammatory pathology of OA. Besides subchondral bone, the authors propose an alternative passage of mast cells invasion in osteophytes, wherein OA SF was found to be necessary and sufficient for maturation of mast cell precursor into effector cells.

Transforming growth factor beta isoforms (TGF-β₁, TGF-β₂, and TGF-β₃) most likely play a role in bone physiology, but little is known about their relative importance in normal as well as in heterotopic bone. This study focused on possible differences in the localization and relative content of different TGF beta isoforms in heterotopic ossifications (HO) by comparing HOs, which have developed less than 17 months (immature HOs) with those developed 3–9 years (mature HOs). The HOs were harvested after total hip arthroplasty (THA) during revision surgery. The HO samples were decalcified, embedded in paraffin and sectioned. Azan staining was used to evaluate histological structure of the ossifications and immunohistochemical analysis was performed to estimate the localization of three TGF beta isoforms in the HOs. Comparison of different TGF beta isoforms in the immature and the mature ossifications showed that the content of TGF-β₂ was decreased by almost three times in the mature HO as compared to the immature HO (p = 0.0064). The proportions of other isoforms in HOs did not differ significantly. This study shows that the relative importance of TGF betas change with HO development.

The aim of this study was to compare different antigen retrieval methods to improve the outcome of immunohistochemistry (IHC) performed on osteoarthritic (OA) cartilage obtained from total knee replacement operation. A voluminous and dense extracellular matrix of articular cartilage inhibits antibody penetration, and therefore, proteins present at low concentrations and masked during fixation may need antigen retrieval to enhance an IHC outcome. We focused on the IHC detection of a minor but diagnostically promising cartilage glycoprotein, CILP-2 (cartilage intermediate layer protein 2), to demonstrate the effect of four different protocols: (1) heat-induced epitope retrieval (HIER), (2) proteolytic-induced epitope retrieval applying proteinase K and hyaluronidase (PIER), (3) HIER combined with PIER, and (4) no antigen retrieval (control). A semi-quantitative staining assessment based on the CILP-2 staining extent was applied. Out of the tested antigen retrieval protocols, the best CILP-2 IHC staining results were achieved by PIER. Combining PIER with HIER did not improve CILP-2 staining in the given experimental setting. Rather the opposite, the application of heat reduced the positive effect of PIER on CILP-2 staining and resulted in the frequent detachment of sections from the slides. Our findings emphasize the need for proper adaptation of antigen retrieval protocols for IHC to maximize the quantitative evaluation of minor matrix proteins in OA articular cartilage samples.

Pathological cleavage of type II collagen (Col2) and generation of Col2 neoepitopes can serve as useful molecular markers of the progression of osteoarthritis (OA). One of such potential biomarkers is type II collagen neoepitope C2C. The aim of this study was to correlate the degree of articular cartilage damage in OA patients with C2C expression in histological samples of tissues removed during total knee replacement. Cartilage samples were obtained from 27 patients ranging in age from 55 to 66 years. In each patient, medial and lateral tibia plateau samples were analyzed according to the OARSI histopathology grading system. The C2C expression was evaluated on histological slides by semi-quantitative analysis using ImageJ Fiji 2.14.0 software. Spearman’s rank correlation analysis revealed a positive weak correlation (rho = 0.289, p = 0.0356) between the histological grade of tissue damage and the percentage of C2C staining. In addition, a highly significant positive correlation (rho = 0.388, p = 0.0041) was discovered between the osteoarthritis score (combining the histological grade of damage with the OA macroscopic stage) and the percentage of C2C staining in the samples. The C2C expression was detected in all the regions of the articular cartilage (i.e., the superficial zone, mid zone, deep zone and tidemark area, and the zone of calcified cartilage). Our findings imply that local expression of C2C correlates with the articular cartilage damage in OA-affected knees. This confirms that C2C can be used as a prospective marker for assessing pathological changes in the OA course and OA clinical trials.

Pathological cleavage of collagen type II (Col2) and generation of Col2 neoepitopes can be good molecular markers of progression of osteoarthritis (OA). One of such potential biomarkers is collagen type II neoepitope C2C. The aim of this study was to correlate the degree of articular cartilage damage in OA patients with C2C expression in histological samples of tissues removed during total knee replacement. Cartilage samples were obtained from 27 patients aged from 55 to 66. In each patient medial and lateral tibia plateau samples were analyzed according to the OARSI histopathology grading system. C2C expression was evaluated on histological slides by semi-quantitative analysis using ImageJ Fiji software. Spearman´s rank correlation analysis showed significant positive correlation (rho=0.289, P=0.0356) between the histological grade of tissue damage and the percentage of C2C staining. In addition, highly significant positive correlation (rho=0.388, P=0.0041) was found between osteoarthritis score (combining the histological grade of damage with the OA macroscopic stage) and the percentage of C2C staining in the samples. The C2C expression was found in all the regions of the articular cartilage (i.e. the superficial zone, mid zone, deep zone and tidemark area and the zone of calcified cartilage). Our study suggests that local expression of C2C correlates with the cartilage damage in the knee affected by OA. This further assures that C2C can be a perspective marker for evaluation of pathological processes in OA course and OA clinical trials.